The present invention relates to cationic polymer-nucleic acid compounds which have use in the delivery of nucleic acid to cells in biological systems, for instance in in vitro cell transfection research. The invention also relates to methods of making such compounds and potentially to gene therapy using such compounds.
The control of living processes is mediated through nucleic acids. Nucleic acids encode proteins which, as enzymes, hormones and other regulatory factors, carry out the processes which enable living organisms to function. Nucleic acids also encode for regulatory sequences which control the expression of proteins.
Because of its central role in living organisms, nucleic acids make an ideal therapeutic target. It is thought that many diseases could be controlled by the manipulation of nucleic acids in living organisms.
The key factor limiting therapies based on nucleic acid manipulation is the ability to deliver nucleic acids to the appropriate compartment of the cells. Nucleic acids are fragile molecules which are highly negatively charged (one negative charge per phosphate group) and which are readily cleaved by nucleases present both in extracellular fluids and intracellular compartments. As a highly charged molecule it will not cross the lipid membranes surrounding the cell, nor can it readily escape from endosomal compartments involved in the uptake of macromolecules into cells. Even RNAi molecules, although smaller in molecular weight, show significant problems of stability and uptake.
Cationic lipid formulations suffer from a number of shortcomings. The lipids used in these formulations are often toxic to cells, and their use as delivery vehicles for nucleic acid to cells can be limited by the toxicity of this component. Cationic lipid formulations are also unstable and have a relatively short shelf life. The short shelf life is at least partly due to the tendency of these formulations to aggregate. Furthermore, lipid formulations are generally expensive.
The use of cationic polymers overcomes some, but not all, of the problems associated with cationic lipid formulations. Polycationic polymers are, however, generally cytotoxic although some cationic polymers with lower toxicity have been reported. Cationic polymers are generally cheap to produce, and do not have the shelf life problems associated with cationic lipids.
Cationic polymers are very efficient at condensing nucleic acids into a small volume and at protecting nucleic acids from degradation by serum nucleases. Interaction is through an equilibrium reaction in which adjustment of the environmental conditions, (salt concentration, pH, molecular weight of each of the polymers) will affect the composition and form of the complexes.
In the formation of the toroids, the processes of condensation of nucleic acids and aggregation of particles are competing, so that these systems tend to be unstable with time and form larger aggregates. This is influenced by the charge ratio of the complexes, and can be reduced by using an excess of one of the components. Generally such complexes are, therefore, made with an excess of polymer, although similar complexes with an excess of nucleic acids also have some favorable properties.
The lack of efficiency of cationic polymer-nucleic acid delivery systems may relate to the efficiency with which they can be taken up into cells, and with which they can escape from the endosomal compartment of the cell, into the cytoplasm. For this reason there has been much research into incorporating ligands and other biologically-active molecules which recognize cell surface receptors involved in endocytosis, and into the use of molecules, such as amphipathic peptides, which can disrupt endosomal membranes.
Cationic polyamines such as polyethylenimine (PEI), poly(L-lysine), polyamidoamines, chitosan, poly(amino ester)s and polyacrylates have been widely investigated as nucleic acid delivery vehicles.
In comparison to cationic polyacrylates, which have ester linkages between the backbone and side groups, there has been less attention directed towards the development of cationic polyacrylamides. This may be due to the perception that acrylamides are less degradable under physiological conditions owing to the more stable amide bond between the side group and polymer backbone. A lack of degradability has often been cited as a possible issue with respect to toxicity and safety.
It is an object of the invention to overcome at least some of the above problems.